Submitted by:
ID1: 320983216 Name1: Maxim Kolchinsky
ID2: 321340580 Name2: Anna Romanov

The Biology

The data for this lesson comes from:
> Saigi et al. “MET-Oncogenic and JAK2-Inactivating Alterations Are Independent Factors That Affect Regulation of PD-L1 Expression in Lung Cancer” PLoS ONE. 2018 Jun 13;9(6):e99625. PMID: 24926665.

Purpose: The blockade of immune checkpoints such as PDL1 and PD-1 is being exploited therapeutically in several types of malignancies. Here, we aimed to understand the contribution of the genetics of lung cancer to the ability of tumor cells to escape immunosurveillance checkpoints. Experimental Design: More than 150 primary non-small cell lung cancers, including pulmonary sarcomatoid carcinomas, were tested for levels of the HLA-I complex, PD-L1, tumor-infiltrating CD8þ lymphocytes, and alterations in main lung cancer genes. Correlations were validated in cancer cell lines using appropriate treatments to activate or inhibit selected pathways. We also performed RNA sequencing to assess changes in gene expression after these treatments. Results: MET-oncogenic activation tended to associate with positive PD-L1 immunostaining, whereas STK11 mutations were correlated with negative immunostaining. In MET-altered cancer cells, MET triggered a transcriptional increase of PD-L1 that was independent of the IFNgmediated JAK/STAT pathway. The activation of MET also upregulated other immunosuppressive genes (PDCD1LG2 and SOCS1) and transcripts involved in angiogenesis (VEGFA and NRP1) and in cell proliferation. We also report recurrent inactivating mutations in JAK2 that co-occur with alterations in MET and STK11, which prevented the induction of immunoresponse-related genes following treatment with IFNg. Conclusions: We show that MET activation promotes the expression of several negative checkpoint regulators of the immunoresponse, including PD-L1. In addition, we report inactivation of JAK2 in lung cancer cells that prevented the response to IFNg. These alterations are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors

Data

This data was downloaded from GEO (GSE:GSE109720)

Import count data and metadata

library(readr)
library(dplyr)
library(ggplot2)

rawcounts <- read_csv("data/lung_counts.csv")
metadata <-  read_csv("data/lung_metadata.csv")

rawcounts
metadata

Clustering - k-means

library(tibble)
library(tidyr)
# a plotting function
plot_heatmap <- function(km) {
  centers <- km$centers %>%
      tbl_df() %>%
      rownames_to_column('Cluster') %>%
      gather(Sample, value, -Cluster) %>%
      mutate(
          Cluster = factor(Cluster),
          Sample = factor(Sample)
      )
   ggplot(centers,aes(Sample,Cluster)) + geom_tile(aes(fill=value)) + geom_text(aes(label = round(value, 1)), angle=90, size=4) + theme(axis.text.x=element_text(angle=90, vjust=0.5, hjust=0, size=12))
}

# cluster the genes
rawcounts_RN<- data.frame(rawcounts, row.names=1)
km <- kmeans(rawcounts_RN,5)
km$centers
##        AE1148      AE1149      AE1150     AE1151      AE1152      AE1153
## 1    204.3794    260.8659    184.8346    225.175    226.5053    263.4797
## 2  31052.4260  42212.6686  27571.1893  34696.260  32887.3373  41325.6095
## 3 261075.5000 355397.5000 249188.5000 298081.500 312029.0000 330232.5000
## 4 101059.4762 147734.7143  89713.5238 117618.429 120754.0000 151602.5238
## 5   6589.0833   8841.2332   6127.4839   7358.684   7346.2171   8890.0477
##        AE1154      AE1155      AE1156      AE1157      AE1158     AE1159
## 1    177.6818    222.9686    196.5011    232.7849    189.8281    212.521
## 2  26505.6568  33457.2663  29596.4379  37330.6331  30042.5799  32418.935
## 3 222399.5000 302143.5000 258949.5000 295158.0000 257717.0000 284918.500
## 4  90998.6190 124462.2857 104368.4286 134149.9048 106395.0952 117189.476
## 5   5868.3233   7272.5940   6356.2941   8001.1913   6352.8116   7040.299
##       AE1160      AE1161      AE1162      AE1163      AE1164      AE1165
## 1    242.584    180.0566    253.0504    219.7236    191.4948    214.1561
## 2  35868.917  27201.2367  34315.4142  31814.4260  29037.9941  29120.4734
## 3 413308.500 281465.5000 419312.0000 335008.5000 264320.5000 344465.0000
## 4 143324.762 104374.0952 132841.0000 122184.8571 107568.4286 112637.7143
## 5   7666.075   5913.3574   7290.1723   6847.2006   6203.9289   6185.2936
##        AE1166      AE1167      AE1168      AE1169      AE1170      AE1171
## 1    241.4646    198.3505    238.9257    253.7209    189.6234    218.4082
## 2  34757.7219  29575.0533  32625.8402  36791.6982  27468.7456  31881.7219
## 3 384117.5000 304171.0000 421639.5000 605629.0000 430845.0000 507135.5000
## 4 135000.0476 111633.0476 126291.2857 169666.4762 124785.2381 141406.8095
## 5   7557.6928   6488.0501   6934.8676   7469.2434   5595.4722   6493.8734
##        AE1172      AE1173      AE1174     AE1175      AE1176      AE1177
## 1    185.7504    226.5967    210.5955    222.858    183.9239    211.8739
## 2  27203.1006  33370.0118  31380.9112  29166.260  24118.6923  28170.8698
## 3 422003.5000 530275.0000 492796.5000 382136.500 308577.5000 364062.5000
## 4 117254.7619 146959.6667 137927.1429 127871.333 104154.3333 123308.0952
## 5   5550.7936   6760.0141   6311.3476   6946.255   5771.8062   6654.9367
##        AE1178      AE1179      AE1180
## 1    165.6252    178.0214    225.1644
## 2  21687.6036  23821.4024  29880.8935
## 3 280669.0000 308508.0000 400801.0000
## 4  94938.3333 104582.6190 133835.4286
## 5   5181.0136   5586.3374   7055.9640
table(km$cluster)
## 
##     1     2     3     4     5 
## 56101   169     2    21  2054
plot_heatmap(km)

# apply log scaling
rawcounts_RN
rawcounts_log = log10(1+rawcounts_RN)
km <- kmeans(rawcounts_log,5)
km$centers
##       AE1148     AE1149     AE1150     AE1151     AE1152     AE1153
## 1 1.83702486 1.90449010 1.80528135 1.87937096 1.91101067 1.93563878
## 2 2.81040539 2.89376012 2.74982450 2.84936858 2.84983522 2.88789617
## 3 0.71791421 0.78602626 0.72984850 0.76302505 0.80766282 0.82175669
## 4 3.53276033 3.64441691 3.48151022 3.57701118 3.57459879 3.63265423
## 5 0.03383464 0.04070399 0.03671669 0.03778014 0.04129412 0.04544332
##       AE1154     AE1155     AE1156     AE1157     AE1158     AE1159
## 1 1.78006310 1.89533169 1.87809325 1.88631317 1.83941134 1.88495685
## 2 2.70968337 2.83817649 2.79121332 2.84003298 2.75247788 2.81786796
## 3 0.71051633 0.78979111 0.78077052 0.78193158 0.75910351 0.77910880
## 4 3.44101008 3.56260329 3.51018120 3.59660666 3.47847058 3.55258537
## 5 0.03525637 0.04041237 0.03816456 0.03878697 0.03846926 0.04042839
##       AE1160     AE1161     AE1162     AE1163     AE1164    AE1165
## 1 1.72482441 1.57883609 1.78440280 1.69279157 1.60659796 1.7010291
## 2 2.85287266 2.70356689 2.87962990 2.81338935 2.73439810 2.8057634
## 3 0.77984051 0.67734644 0.83405850 0.75250277 0.68961210 0.7527325
## 4 3.60604751 3.47295929 3.59456780 3.55882952 3.49697270 3.5233552
## 5 0.04228286 0.03482157 0.04934553 0.04090791 0.03483581 0.0403937
##       AE1166     AE1167    AE1168     AE1169     AE1170     AE1171
## 1 1.72148517 1.61817379 1.7524813 1.86774760 1.73868498 1.80511054
## 2 2.84957931 2.74917160 2.8518106 2.86213862 2.73553353 2.79738975
## 3 0.76919789 0.68795606 0.7883144 0.97743341 0.86818522 0.92806724
## 4 3.60198434 3.51694702 3.5721743 3.58005983 3.45251003 3.51893319
## 5 0.04050045 0.03600679 0.0450553 0.07469409 0.05966218 0.06738637
##       AE1172     AE1173     AE1174     AE1175     AE1176     AE1177
## 1 1.72950970 1.81404225 1.78408763 1.66421354 1.57459353 1.63890307
## 2 2.72540850 2.81188997 2.78091975 2.77071334 2.68425641 2.74768434
## 3 0.85480390 0.93153736 0.91315560 0.72290179 0.65421636 0.71199777
## 4 3.44953976 3.53572936 3.50371977 3.54632120 3.46398147 3.52648075
## 5 0.05931824 0.06742127 0.06470466 0.04220111 0.03480657 0.04185064
##       AE1178     AE1179     AE1180
## 1 1.53851602 1.56294388 1.66357207
## 2 2.64155117 2.67351055 2.77413859
## 3 0.63276763 0.63743320 0.72606404
## 4 3.41852004 3.45101801 3.55283952
## 5 0.03489193 0.03510521 0.04234835
table(km$cluster)
## 
##     1     2     3     4     5 
##  3986  5706  6081  4922 37652
plot_heatmap(km)

# load DE gene list
DE_res <- read_csv("data/sigresults.csv")
DE_genes <- DE_res$row
nrow(DE_genes)
## NULL
# filter differentially expressed genes
dat_filtered = rawcounts_log[DE_genes,]
# utils::View(dat_filtered) #show all rows (sanity test)
dat_filtered
km <- kmeans(dat_filtered,5)
km$centers
##     AE1148    AE1149    AE1150    AE1151    AE1152    AE1153    AE1154
## 1 3.727994 3.8458092 3.6789046 3.7737672 3.7712481 3.8353965 3.6410604
## 2 1.675386 1.7422638 1.6510038 1.7196991 1.7529987 1.7768871 1.6264149
## 3 0.765454 0.8311759 0.7725323 0.8022541 0.8501813 0.8652445 0.7607886
## 4 2.462767 2.5365933 2.4048882 2.5013205 2.5130691 2.5449090 2.3716124
## 5 3.092749 3.1880818 3.0341405 3.1334550 3.1313785 3.1766484 2.9912540
##      AE1155    AE1156    AE1157    AE1158    AE1159   AE1160    AE1161
## 1 3.7607523 3.7065729 3.8005181 3.6777912 3.7524569 3.806034 3.6761759
## 2 1.7360776 1.7220732 1.7293267 1.6861379 1.7252916 1.555795 1.4185047
## 3 0.8368235 0.8265967 0.8244078 0.8075869 0.8277963 0.794030 0.6944175
## 4 2.5022342 2.4616528 2.4920190 2.4193247 2.4803901 2.458831 2.2998114
## 5 3.1179479 3.0678641 3.1348971 3.0303171 3.1023517 3.157674 3.0154272
##      AE1162    AE1163    AE1164    AE1165    AE1166   AE1167    AE1168
## 1 3.7855799 3.7573965 3.6983879 3.7145398 3.8002028 3.718978 3.7639160
## 2 1.6178264 1.5275811 1.4457768 1.5358955 1.5550413 1.454924 1.5847437
## 3 0.8437592 0.7699717 0.7109858 0.7699636 0.7789845 0.707016 0.8029044
## 4 2.5035578 2.4215862 2.3286569 2.4247827 2.4515474 2.342389 2.4701160
## 5 3.1672278 3.1142560 3.0444352 3.0951235 3.1562218 3.061380 3.1431866
##     AE1169   AE1170   AE1171   AE1172   AE1173   AE1174    AE1175
## 1 3.767829 3.639808 3.708035 3.639053 3.725154 3.693315 3.7348540
## 2 1.687502 1.557819 1.624723 1.550872 1.634168 1.606828 1.4788446
## 3 1.161839 1.047954 1.115588 1.034681 1.117392 1.097845 0.7847045
## 4 2.501827 2.375108 2.439605 2.363916 2.449952 2.419957 2.3747731
## 5 3.161516 3.034307 3.096639 3.026127 3.112850 3.080786 3.0987064
##      AE1176    AE1177    AE1178    AE1179    AE1180
## 1 3.6533539 3.7155625 3.6073938 3.6401817 3.7419750
## 2 1.3898792 1.4576452 1.3570359 1.3815305 1.4811532
## 3 0.7185449 0.7670533 0.6898521 0.6939588 0.7796293
## 4 2.2852559 2.3481420 2.2440608 2.2728393 2.3748266
## 5 3.0145756 3.0772103 2.9698454 3.0019990 3.1036586
table(km$cluster)
## 
##    1    2    3    4    5 
## 2659 3066 3832 3205 5195
plot_heatmap(km)

# a plotting function
plot_heatmap <- function(km) {
  centers <- km$centers %>%
      tbl_df() %>%
      rownames_to_column('Cluster') %>%
      gather(Gene, value, -Cluster) %>%
      mutate(
          Cluster = factor(Cluster),
          Gene = factor(Gene)
      )
   ggplot(centers,aes(Cluster,Gene)) + geom_tile(aes(fill=value)) + theme(axis.text.x=element_text(angle=0, vjust=0.5, hjust=0, size=12)) + theme(axis.text.y=element_blank())
}

#filter example
rawcounts_filtered <- rawcounts_RN %>% sample_n(500) 
# cluster the samples
km <- kmeans(t(rawcounts_filtered),3) 
# t function turns samples into rows and genes into columns, this way genes are the features and samples are clustered
km$centers
##   ENSG00000105137.12 ENSG00000188729.6 ENSG00000173705.8 ENSG00000253339.1
## 1          529.90909        0.09090909          195.4545              0.00
## 2           74.16667        0.91666667          645.5833              1.75
## 3          128.20000        0.20000000           14.4000              0.00
##   ENSG00000134259.3 ENSG00000258420.1 ENSG00000231706.2 ENSG00000281394.1
## 1          180.7273                 0                 0                 0
## 2            5.7500                 0                 0                 0
## 3           10.9000                 0                 0                 0
##   ENSG00000204231.10 ENSG00000263603.1 ENSG00000242259.8 ENSG00000247095.2
## 1           1204.545          1.181818          342.9091         139.81818
## 2           1114.833          2.416667          718.1667          57.83333
## 3           2220.500          5.200000          435.9000          61.90000
##   ENSG00000235862.2 ENSG00000166313.18 ENSG00000071242.11
## 1                 0              41.00           1000.636
## 2                 0              25.75            864.500
## 3                 0             152.20           1134.100
##   ENSG00000243854.3 ENSG00000103363.14 ENSG00000252035.1 ENSG00000281566.2
## 1         0.3636364           4620.545                 0           0.00000
## 2         0.4166667           3500.000                 0          15.33333
## 3         1.0000000           3326.600                 0           0.00000
##   ENSG00000233121.1 ENSG00000189348.6 ENSG00000241622.1 ENSG00000156273.15
## 1        0.18181818                 0        0.00000000          2523.7273
## 2        0.08333333                 0        0.08333333           975.3333
## 3        0.00000000                 0        0.10000000          1419.1000
##   ENSG00000227630.3 ENSG00000267275.1 ENSG00000121380.12 ENSG00000228651.1
## 1          11.00000               0.0           26.18182        0.00000000
## 2          10.33333               0.0            1.50000        0.08333333
## 3          10.00000               0.2           89.30000        0.00000000
##   ENSG00000233117.2 ENSG00000278771.1 ENSG00000104970.10 ENSG00000234110.1
## 1         8.6363636          1.363636         0.00000000                 0
## 2         0.1666667         11.416667         0.08333333                 0
## 3       144.7000000          7.000000         0.00000000                 0
##   ENSG00000182218.9 ENSG00000233693.1 ENSG00000115461.4 ENSG00000164713.9
## 1          25.00000         0.6363636          6192.091          932.7273
## 2          33.58333         0.9166667          5020.500         4405.7500
## 3           1.40000         0.1000000           318.300         1477.6000
##   ENSG00000249433.1 ENSG00000240354.1 ENSG00000204622.11 ENSG00000244932.2
## 1                 0                 0           10.09091                 0
## 2                 0                 0            5.00000                 0
## 3                 0                 0           22.30000                 0
##   ENSG00000239820.3 ENSG00000255149.1 ENSG00000137809.16
## 1        0.18181818                 0         121.090909
## 2        0.08333333                 0           5.666667
## 3        0.50000000                 0         344.200000
##   ENSG00000100427.15 ENSG00000160213.6 ENSG00000221858.3 ENSG00000264176.1
## 1          0.2727273          5661.545                 0        0.27272727
## 2          5.6666667          4871.083                 0        0.08333333
## 3          0.3000000          2072.300                 0        0.80000000
##   ENSG00000197444.9 ENSG00000258944.1 ENSG00000277630.4 ENSG00000237823.1
## 1          20.63636          5.727273                 0                 0
## 2          18.41667          5.250000                 0                 0
## 3           8.60000          4.500000                 0                 0
##   ENSG00000254019.1 ENSG00000073331.17 ENSG00000115474.6 ENSG00000122728.6
## 1         0.0000000           510.6364               0.0              2.00
## 2         0.1666667           486.2500               0.0              4.75
## 3         0.0000000          1281.7000               9.3              2.00
##   ENSG00000204456.4 ENSG00000196578.4 ENSG00000167751.12 ENSG00000277418.1
## 1                 0        0.00000000         0.09090909                 0
## 2                 0        0.08333333         1.16666667                 0
## 3                 0        0.00000000         1.90000000                 0
##   ENSG00000204628.11 ENSG00000169900.7 ENSG00000233214.1 ENSG00000213977.7
## 1           38426.36        0.00000000        0.09090909          10491.36
## 2           29081.25        0.08333333        0.00000000           2322.00
## 3           28427.90        0.00000000        0.00000000           5111.30
##   ENSG00000230709.1 ENSG00000198756.10 ENSG00000197915.5 ENSG00000235045.2
## 1        0.09090909           52.00000         0.5454545        0.27272727
## 2        0.08333333           10.33333         0.4166667        0.08333333
## 3        0.00000000           17.00000         0.1000000        0.00000000
##   ENSG00000254630.1 ENSG00000264128.2 ENSG00000238007.1 ENSG00000162722.8
## 1        0.00000000                 0                 0               0.0
## 2        0.08333333                 0                 0             149.5
## 3        0.20000000                 0                 0               0.1
##   ENSG00000224374.1 ENSG00000254528.7 ENSG00000105173.13 ENSG00000171747.8
## 1                 0           0.00000           958.6364          2.636364
## 2                 0          43.58333          1644.4167          6.750000
## 3                 0           0.20000           305.6000          2.700000
##   ENSG00000225171.2 ENSG00000173452.13 ENSG00000198887.8 ENSG00000226670.1
## 1          1.909091         0.00000000          2455.727                 0
## 2          2.166667         0.08333333          2219.500                 0
## 3          1.100000         0.00000000          4153.500                 0
##   ENSG00000175414.6 ENSG00000267409.1 ENSG00000236242.1 ENSG00000225449.3
## 1          9.727273                 0          6.818182           0.00000
## 2        114.333333                 0          3.416667          15.91667
## 3          1.900000                 0          0.300000           0.00000
##   ENSG00000270669.1 ENSG00000077942.18 ENSG00000256136.1
## 1                 0           3679.636                 0
## 2                 0           2487.667                 0
## 3                 0            322.200                 0
##   ENSG00000175063.16 ENSG00000210174.1 ENSG00000263321.1 ENSG00000243011.3
## 1           5150.727                 0                 0                 0
## 2           2794.750                 0                 0                 0
## 3           3831.600                 0                 0                 0
##   ENSG00000219553.2 ENSG00000170967.4 ENSG00000223546.6 ENSG00000131142.13
## 1         0.3636364         0.0000000         140.18182         0.09090909
## 2         0.9166667         0.1666667          96.33333         0.08333333
## 3         0.2000000         0.0000000         172.90000         0.20000000
##   ENSG00000226950.6 ENSG00000141040.14 ENSG00000270837.1 ENSG00000235661.3
## 1          820.0909            0.00000                 0                 0
## 2         2041.1667           90.08333                 0                 0
## 3         1919.6000           38.70000                 0                 0
##   ENSG00000212316.1 ENSG00000277196.4 ENSG00000225603.3 ENSG00000253833.1
## 1                 0          145.6364         0.3636364        0.09090909
## 2                 0          327.1667         0.1666667        0.91666667
## 3                 0           18.1000         0.4000000        1.00000000
##   ENSG00000156471.12 ENSG00000227050.1 ENSG00000269320.1 ENSG00000279874.1
## 1           4656.909              0.00                 0                 0
## 2           4463.417              0.25                 0                 0
## 3           6881.000              0.40                 0                 0
##   ENSG00000228496.1 ENSG00000275107.1 ENSG00000232406.6 ENSG00000179899.8
## 1               0.0                 0        0.09090909          31.45455
## 2               0.5                 0        0.66666667          89.83333
## 3               0.0                 0        3.50000000         168.20000
##   ENSG00000219074.1 ENSG00000164220.6 ENSG00000267418.1 ENSG00000212335.1
## 1                 0          20.27273                 0                 0
## 2                 0         116.50000                 0                 0
## 3                 0           1.00000                 0                 0
##   ENSG00000207750.1 ENSG00000234385.1 ENSG00000250714.3 ENSG00000169696.15
## 1                 0                 0          5.818182           798.7273
## 2                 0                 0          2.416667          1146.2500
## 3                 0                 0          3.200000           869.5000
##   ENSG00000220695.1 ENSG00000227088.1 ENSG00000189366.9 ENSG00000211941.3
## 1         0.7272727                 0         497.63636                 0
## 2         0.4166667                 0          47.58333                 0
## 3         0.6000000                 0         104.40000                 0
##   ENSG00000229722.1 ENSG00000261703.1 ENSG00000123144.10 ENSG00000280081.3
## 1                 0                 0           2842.636          0.000000
## 2                 0                 0           1794.000          2.666667
## 3                 0                 0           2128.100          0.000000
##   ENSG00000271616.1 ENSG00000207515.1 ENSG00000240625.3 ENSG00000128602.9
## 1         0.0000000                 0              0.00          82.90909
## 2         0.1666667                 0              0.25         436.00000
## 3         0.1000000                 0              0.60           8.80000
##   ENSG00000088247.17 ENSG00000183486.12 ENSG00000147885.4
## 1           6882.909           3099.636                 0
## 2           6795.167             10.250                 0
## 3           9260.200            707.600                 0
##   ENSG00000266265.2 ENSG00000257504.1 ENSG00000161920.9 ENSG00000284116.1
## 1          1.363636              0.00          507.6364          98.54545
## 2          8.000000              0.25          394.0000          23.83333
## 3          0.100000              0.00          381.4000         126.40000
##   ENSG00000233489.1 ENSG00000164934.13 ENSG00000252716.1 ENSG00000249041.1
## 1                 0           3858.818                 0                 0
## 2                 0           5296.500                 0                 0
## 3                 0           3655.100                 0                 0
##   ENSG00000250424.4 ENSG00000283154.2 ENSG00000272827.1 ENSG00000271475.1
## 1         0.2727273          899.3636                 0        0.00000000
## 2         0.9166667          401.2500                 0        0.08333333
## 3         0.1000000          768.5000                 0        0.10000000
##   ENSG00000268555.1 ENSG00000235028.3 ENSG00000154265.15 ENSG00000187870.7
## 1        0.18181818         0.5454545           580.7273          0.000000
## 2        0.08333333         0.4166667          1245.1667          5.083333
## 3        0.00000000         0.0000000           129.6000          5.000000
##   ENSG00000245146.6 ENSG00000254136.1 ENSG00000188039.13 ENSG00000202217.1
## 1          81.18182                 0           40.45455                 0
## 2         103.16667                 0           83.50000                 0
## 3         109.00000                 0          107.70000                 0
##   ENSG00000223965.2 ENSG00000232463.1 ENSG00000255234.5 ENSG00000176160.10
## 1                 0                 0         0.9090909          0.2727273
## 2                 0                 0         4.0833333          0.2500000
## 3                 0                 0         1.0000000          0.4000000
##   ENSG00000122136.13 ENSG00000262899.1 ENSG00000256994.1
## 1               0.00        0.09090909        0.00000000
## 2               0.25        0.00000000        0.08333333
## 3               0.10        0.00000000        0.00000000
##   ENSG00000099715.14 ENSG00000241095.1 ENSG00000211863.1 ENSG00000234812.1
## 1                  0         0.8181818                 0                 0
## 2                  0         2.1666667                 0                 0
## 3                  0         1.3000000                 0                 0
##   ENSG00000006611.15 ENSG00000241612.1 ENSG00000133818.13
## 1           0.000000         0.7272727           5603.364
## 2           3.333333         0.4166667           1048.167
## 3           0.000000         0.5000000           2631.000
##   ENSG00000276707.1 ENSG00000105583.9 ENSG00000267909.2 ENSG00000189184.11
## 1                 0           2066.00          1.090909        261.5454545
## 2                 0           1508.25          6.416667          0.1666667
## 3                 0           1565.40          0.100000         26.8000000
##   ENSG00000170502.12 ENSG00000105254.11 ENSG00000231787.4
## 1           759.2727           1246.455               0.0
## 2          1554.9167           1296.833               0.0
## 3          1378.5000           1586.800               0.7
##   ENSG00000241003.1 ENSG00000235797.2 ENSG00000008517.16 ENSG00000266097.1
## 1         0.1818182        0.09090909           902.3636                 0
## 2         0.8333333        0.08333333            19.5000                 0
## 3         0.6000000        0.00000000           861.9000                 0
##   ENSG00000229056.2 ENSG00000199815.2 ENSG00000201014.1 ENSG00000234055.1
## 1          5.454545                 0                 0                 0
## 2         39.833333                 0                 0                 0
## 3          0.500000                 0                 0                 0
##   ENSG00000239215.1 ENSG00000169306.9 ENSG00000223921.1 ENSG00000239595.3
## 1                 0        21.0909091        0.00000000                 0
## 2                 0         0.3333333        0.08333333                 0
## 3                 0       304.2000000        0.00000000                 0
##   ENSG00000103351.12 ENSG00000236325.1 ENSG00000255977.1 ENSG00000225905.1
## 1           769.6364                 0        0.00000000         1.2727273
## 2           442.1667                 0        0.08333333         0.3333333
## 3          2006.8000                 0        0.00000000         0.2000000
##   ENSG00000140688.16 ENSG00000261335.1 ENSG00000249863.2 ENSG00000271644.1
## 1           2811.273          7.818182          12.45455         0.1818182
## 2           2981.667         26.583333          57.91667         0.2500000
## 3           2787.800         23.700000           8.30000         0.3000000
##   ENSG00000254556.1 ENSG00000164161.9 ENSG00000235082.2 ENSG00000182752.9
## 1              0.00          13.18182          8.272727          149.3636
## 2              0.25           0.75000          4.416667          259.5833
## 3              3.20           1.60000          2.400000           53.4000
##   ENSG00000144426.18 ENSG00000230982.1 ENSG00000137959.15
## 1           754.4545                 0           3089.727
## 2           982.1667                 0            139.000
## 3           557.4000                 0           3491.500
##   ENSG00000187857.4 ENSG00000159399.9 ENSG00000227023.1 ENSG00000253523.1
## 1                 0          5701.182                 0                 0
## 2                 0          1531.583                 0                 0
## 3                 0          1710.100                 0                 0
##   ENSG00000252457.1 ENSG00000275002.1 ENSG00000112812.15 ENSG00000275484.1
## 1                 0                 0           342.9091          8.272727
## 2                 0                 0           281.6667         16.833333
## 3                 0                 0           580.9000          8.800000
##   ENSG00000218739.9 ENSG00000176087.14 ENSG00000254669.1 ENSG00000198367.3
## 1          987.0909           3883.818                 0                 0
## 2         1894.5833           3157.083                 0                 0
## 3         1704.8000            759.000                 0                 0
##   ENSG00000218472.2 ENSG00000104290.10 ENSG00000224677.1 ENSG00000230852.1
## 1               0.0           138.2727                 0                 0
## 2               0.0           459.7500                 0                 0
## 3               0.3           310.4000                 0                 0
##   ENSG00000213731.2 ENSG00000101849.15 ENSG00000261663.1 ENSG00000164778.4
## 1                 0           840.8182          3.363636          0.000000
## 2                 0          3131.2500          3.333333          8.333333
## 3                 0           533.6000          8.900000          0.100000
##   ENSG00000164185.5 ENSG00000272685.1 ENSG00000128607.13 ENSG00000277413.1
## 1          2.636364                 0           1808.455                 0
## 2         18.083333                 0           1434.167                 0
## 3          8.800000                 0           1673.900                 0
##   ENSG00000229595.1 ENSG00000257964.1 ENSG00000115310.17 ENSG00000176230.5
## 1        0.09090909        0.00000000           11159.82                 0
## 2        0.00000000        0.08333333            7028.25                 0
## 3        0.20000000        0.60000000            7664.80                 0
##   ENSG00000283657.1 ENSG00000226338.1 ENSG00000227186.1 ENSG00000164171.10
## 1                 0                 0                 0           13511.45
## 2                 0                 0                 0           11408.67
## 3                 0                 0                 0           22331.70
##   ENSG00000202081.1 ENSG00000229533.1 ENSG00000242727.2 ENSG00000201684.1
## 1                 0                 0                 0         0.6363636
## 2                 0                 0                 0         1.0833333
## 3                 0                 0                 0         0.7000000
##   ENSG00000261418.1 ENSG00000260565.6 ENSG00000274029.1 ENSG00000219951.4
## 1        0.09090909          814.7273         0.2727273          1.181818
## 2        0.33333333          633.6667         0.0000000          0.500000
## 3        0.70000000         1072.8000         0.3000000          0.900000
##   ENSG00000246985.7 ENSG00000044446.11 ENSG00000110077.14
## 1          81.72727           1411.545         0.00000000
## 2          33.16667           1027.417         0.08333333
## 3          43.20000           1140.100         0.20000000
##   ENSG00000233852.1 ENSG00000111186.12 ENSG00000271304.1
## 1              0.00          185.09091                 0
## 2              0.25           16.41667                 0
## 3              0.00           13.10000                 0
##   ENSG00000139793.18 ENSG00000201752.1 ENSG00000165819.11
## 1           1665.818                 0           2447.636
## 2            478.250                 0           2739.333
## 3           2550.600                 0            986.800
##   ENSG00000147381.11 ENSG00000266756.1 ENSG00000176834.13
## 1                0.0                 0           820.3636
## 2            12636.5                 0          1721.5000
## 3                0.1                 0          2341.5000
##   ENSG00000225942.1 ENSG00000226646.1 ENSG00000080824.18 ENSG00000258107.1
## 1        0.09090909                 0           81909.73                 0
## 2        0.00000000                 0           91225.67                 0
## 3        0.00000000                 0           61063.60                 0
##   ENSG00000255146.1 ENSG00000162976.12 ENSG00000265912.1
## 1        0.09090909           509.9091         0.7272727
## 2        0.83333333           572.7500         0.5833333
## 3        0.00000000           145.2000         1.1000000
##   ENSG00000167614.13 ENSG00000201558.1 ENSG00000100867.14
## 1          0.8181818         0.3636364           186.7273
## 2         12.6666667         1.0833333           798.1667
## 3         45.0000000         0.2000000           148.6000
##   ENSG00000224089.3 ENSG00000204687.4 ENSG00000258914.1 ENSG00000267774.2
## 1                 0                 0                 0         6.6363636
## 2                 0                 0                 0         0.1666667
## 3                 0                 0                 0         0.5000000
##   ENSG00000271595.1 ENSG00000211783.3 ENSG00000263821.1 ENSG00000201839.1
## 1                 0                 0                 0                 0
## 2                 0                 0                 0                 0
## 3                 0                 0                 0                 0
##   ENSG00000249348.1 ENSG00000212939.2 ENSG00000236231.1 ENSG00000149634.4
## 1          31.45455        0.00000000                 0          21.18182
## 2          45.41667        0.08333333                 0           4.50000
## 3          36.00000        0.10000000                 0          24.10000
##   ENSG00000197712.11 ENSG00000055211.12 ENSG00000119660.4
## 1           1000.545           1309.818         0.0000000
## 2           1612.250            686.000         0.1666667
## 3            461.600           1284.100         0.0000000
##   ENSG00000241127.7 ENSG00000228734.2 ENSG00000185477.4 ENSG00000253820.1
## 1          885.0000                 0         0.5454545                 0
## 2          273.4167                 0      1647.0833333                 0
## 3          407.0000                 0         0.3000000                 0
##   ENSG00000257472.1 ENSG00000249915.7 ENSG00000180667.10 ENSG00000283491.1
## 1                 0         17560.818           2066.909         0.6363636
## 2                 0          7014.417           1528.583         2.3333333
## 3                 0          2361.700            602.200         2.6000000
##   ENSG00000240583.11 ENSG00000205424.1 ENSG00000124205.15
## 1          110.09091                 0          0.0000000
## 2           25.08333                 0          0.3333333
## 3           13.10000                 0          0.0000000
##   ENSG00000248850.1 ENSG00000203780.10 ENSG00000161904.11
## 1                 0              15.00           2602.545
## 2                 0             169.25           1360.000
## 3                 0             324.00           2604.800
##   ENSG00000213331.4 ENSG00000227882.1 ENSG00000230716.3 ENSG00000143367.15
## 1         0.8181818                 0          10.72727           3694.909
## 2         0.1666667                 0          13.75000           3116.000
## 3         0.2000000                 0           8.90000           1930.000
##   ENSG00000175294.5 ENSG00000104714.13 ENSG00000005513.9 ENSG00000259622.1
## 1         26.000000           441.4545         0.2727273                 0
## 2          5.166667           231.7500         7.2500000                 0
## 3         75.300000           542.8000        14.5000000                 0
##   ENSG00000228252.9 ENSG00000254921.1 ENSG00000146039.10 ENSG00000279378.1
## 1        0.18181818                 0         0.00000000         0.0000000
## 2        0.08333333                 0         0.08333333         0.4166667
## 3        0.50000000                 0         0.00000000         0.0000000
##   ENSG00000161640.15 ENSG00000202224.1 ENSG00000237260.1 ENSG00000269001.2
## 1               0.00                 0         0.1818182         381.54545
## 2               0.25                 0         0.5833333          69.41667
## 3               0.00                 0         0.1000000         216.40000
##   ENSG00000223904.2 ENSG00000166793.10 ENSG00000267096.1 ENSG00000225836.1
## 1               0.0           1.818182         16.636364                 0
## 2               0.0           0.250000          8.666667                 0
## 3               0.1          25.100000         13.800000                 0
##   ENSG00000259848.8 ENSG00000242628.5 ENSG00000223419.1 ENSG00000126261.12
## 1        0.09090909                 0                 0           6035.909
## 2        5.08333333                 0                 0           5496.083
## 3        0.80000000                 0                 0           5254.000
##   ENSG00000182795.12 ENSG00000006607.13 ENSG00000266065.1
## 1           7676.909           1321.545                 0
## 2           1606.750           1895.667                 0
## 3           1432.100           1007.300                 0
##   ENSG00000283047.1 ENSG00000157654.17 ENSG00000252297.1 ENSG00000178082.6
## 1        0.09090909           85.90909                 0          8.818182
## 2      134.50000000           47.16667                 0         11.750000
## 3        0.30000000           78.70000                 0         11.000000
##   ENSG00000258994.1 ENSG00000232989.1 ENSG00000168484.12 ENSG00000232144.1
## 1                 0                 0          1.0909091                 0
## 2                 0                 0          0.4166667                 0
## 3                 0                 0          0.4000000                 0
##   ENSG00000272333.5 ENSG00000283450.1 ENSG00000270058.1 ENSG00000166225.8
## 1          2239.091                 0         0.1818182          778.8182
## 2          3522.917                 0         0.0000000         1600.3333
## 3          2699.400                 0         0.0000000         1252.1000
##   ENSG00000128000.15 ENSG00000240096.1 ENSG00000276493.1 ENSG00000169562.9
## 1           391.5455         0.0000000                 0               0.0
## 2           215.1667         0.1666667                 0               4.5
## 3           946.5000         0.0000000                 0               0.1
##   ENSG00000251670.1 ENSG00000253284.2 ENSG00000269678.1 ENSG00000280739.2
## 1                 0          2.181818                 0          8.727273
## 2                 0          3.250000                 0         16.833333
## 3                 0          5.800000                 0          5.500000
##   ENSG00000255114.1 ENSG00000211877.1 ENSG00000283164.1 ENSG00000225405.3
## 1        0.09090909                 0                 0                 0
## 2        0.16666667                 0                 0                 0
## 3        0.10000000                 0                 0                 0
##   ENSG00000240549.2 ENSG00000224888.4 ENSG00000278413.1 ENSG00000219410.5
## 1                 0          1.909091                 0              0.00
## 2                 0          4.500000                 0              0.75
## 3                 0          0.700000                 0              0.90
##   ENSG00000223740.1 ENSG00000106153.12 ENSG00000115970.18
## 1                 0           15539.55           1505.818
## 2                 0           12946.75           1681.500
## 3                 0            7465.60           1828.300
##   ENSG00000237001.6 ENSG00000274929.1 ENSG00000260720.1 ENSG00000107438.8
## 1                 0         0.9090909                 0         14399.636
## 2                 0         3.2500000                 0          2290.333
## 3                 0         2.1000000                 0          4285.800
##   ENSG00000100916.13 ENSG00000254976.1 ENSG00000111490.12
## 1           632.8182                 0           493.2727
## 2           764.5833                 0          2135.6667
## 3           900.3000                 0           424.0000
##   ENSG00000237997.1 ENSG00000143167.11 ENSG00000225122.1 ENSG00000221139.1
## 1                 0         0.09090909                 0                 0
## 2                 0         6.91666667                 0                 0
## 3                 0         0.40000000                 0                 0
##   ENSG00000167799.9 ENSG00000253361.1 ENSG00000151929.9 ENSG00000271730.1
## 1          148.0000                 0          1591.545         0.0000000
## 2          318.3333                 1          3956.833         0.1666667
## 3           85.1000                 0          1570.300         0.3000000
##   ENSG00000117859.18 ENSG00000207161.1 ENSG00000053438.8 ENSG00000069482.6
## 1           2927.455                 0        0.27272727          97.54545
## 2           3205.250                 0        0.08333333         795.41667
## 3           5472.400                 0        0.00000000          11.90000
##   ENSG00000136250.11 ENSG00000254767.1 ENSG00000258593.2
## 1           7.272727                 0              7.00
## 2          15.916667                 0              8.75
## 3           1.000000                 0             11.30
##   ENSG00000165966.14 ENSG00000231396.2 ENSG00000130775.15
## 1          3.0000000                 0          57.818182
## 2          0.5833333                 0           6.166667
## 3          0.4000000                 0          20.900000
##   ENSG00000153071.14 ENSG00000138074.14 ENSG00000277559.1
## 1          928.27273           2076.636           52.0000
## 2           91.83333           3294.917          118.8333
## 3          855.20000           2772.000          114.1000
##   ENSG00000034152.18 ENSG00000007171.16 ENSG00000260909.1
## 1           4526.455           1.727273                 0
## 2           2584.583           1.333333                 0
## 3           4280.100           1.400000                 0
##   ENSG00000163518.10 ENSG00000271075.1 ENSG00000279458.2 ENSG00000259342.1
## 1                0.0               0.0          2.818182        0.18181818
## 2                0.0               0.0          0.500000        0.08333333
## 3                0.1               0.3          0.800000        0.10000000
##   ENSG00000256385.1 ENSG00000258934.1 ENSG00000271415.1 ENSG00000164199.16
## 1        0.09090909                 0                 0           4.363636
## 2        0.08333333                 0                 0        1921.166667
## 3        0.00000000                 0                 0         352.400000
##   ENSG00000183831.6 ENSG00000254718.6 ENSG00000278351.1 ENSG00000089195.14
## 1          1.545455          1.636364              0.00           4210.545
## 2          3.583333          0.250000              0.25            761.000
## 3          1.900000          1.400000              0.00           1965.300
##   ENSG00000253910.2 ENSG00000223650.1 ENSG00000223519.8 ENSG00000117335.19
## 1          313.4545        0.18181818                 0          10934.636
## 2           20.0000        0.08333333                 0           8149.417
## 3          382.1000        0.00000000                 0           7958.200
##   ENSG00000239280.1 ENSG00000255481.2 ENSG00000266288.1 ENSG00000264755.1
## 1              2.00                 0                 0                 0
## 2              2.25                 0                 0                 0
## 3              3.40                 0                 0                 0
##   ENSG00000214807.2 ENSG00000262319.1 ENSG00000251076.1 ENSG00000207769.1
## 1        0.09090909        0.09090909          7.454545                 0
## 2        0.25000000        2.16666667          1.500000                 0
## 3        0.10000000        1.00000000          1.100000                 0
##   ENSG00000268940.5 ENSG00000275266.1 ENSG00000244480.1 ENSG00000232511.4
## 1          0.000000                 0          7.909091         0.0000000
## 2          3.416667                 0         24.583333         0.1666667
## 3          0.000000                 0         15.400000         0.0000000
##   ENSG00000227835.8 ENSG00000227056.2 ENSG00000213167.3 ENSG00000187118.12
## 1         0.1818182                 0                 0           329.9091
## 2         0.0000000                 0                 0           470.0000
## 3         0.2000000                 0                 0           309.3000
##   ENSG00000202395.1 ENSG00000265089.1 ENSG00000227597.1 ENSG00000206604.1
## 1                 0                 0                 0                 0
## 2                 0                 0                 0                 0
## 3                 0                 0                 0                 0
##   ENSG00000275638.1 ENSG00000266984.1 ENSG00000271917.1 ENSG00000253932.1
## 1         0.8181818                 0        0.09090909                 0
## 2         0.6666667                 0        0.16666667                 0
## 3         1.2000000                 0        0.00000000                 0
##   ENSG00000284690.1 ENSG00000252003.1 ENSG00000254812.1 ENSG00000113558.18
## 1        0.09090909                 0         0.4545455           7737.818
## 2        0.25000000                 0         3.3333333          10746.917
## 3        0.40000000                 0         0.5000000           9339.400
##   ENSG00000234197.1 ENSG00000233885.7 ENSG00000109270.12
## 1                 0          4.363636           1075.091
## 2                 0          4.250000            805.250
## 3                 0          6.000000            804.900
##   ENSG00000091164.12 ENSG00000244131.2 ENSG00000270147.1 ENSG00000280587.1
## 1           1796.364        0.00000000          3.363636          8.818182
## 2           3913.333        0.08333333          4.333333         28.833333
## 3           3834.300        0.00000000          4.500000         18.200000
##   ENSG00000242431.2 ENSG00000224243.1 ENSG00000251066.1 ENSG00000250441.1
## 1        0.00000000                 0          1.636364               0.0
## 2        0.08333333                 0          4.916667               0.0
## 3        0.00000000                 0          3.600000               0.1
##   ENSG00000260555.1 ENSG00000207245.1 ENSG00000252041.1 ENSG00000260280.5
## 1                 0                 0                 0          529.4545
## 2                 0                 0                 0          608.3333
## 3                 0                 0                 0          466.4000
##   ENSG00000237418.1 ENSG00000171606.17 ENSG00000049167.13
## 1        0.81818182          2695.3636           625.0909
## 2        0.08333333           416.4167           383.8333
## 3        1.30000000          1657.4000           426.7000
##   ENSG00000264558.1 ENSG00000254325.2 ENSG00000146250.6 ENSG00000273956.1
## 1         0.0000000        0.09090909          3.818182                 0
## 2         0.1666667        0.00000000         40.750000                 0
## 3         0.1000000        0.00000000         10.900000                 0
##   ENSG00000277184.1 ENSG00000229881.1 ENSG00000255320.1 ENSG00000222990.1
## 1        0.00000000                 0         0.4545455                 0
## 2        0.08333333                 0         0.1666667                 0
## 3        0.10000000                 0         0.4000000                 0
##   ENSG00000160087.20 ENSG00000259607.1 ENSG00000187170.4 ENSG00000248104.1
## 1           1754.364                 0                 0                 0
## 2           2108.583                 0                 0                 0
## 3           1564.300                 0                 0                 0
##   ENSG00000197122.11 ENSG00000267206.5 ENSG00000196313.11
## 1          4273.5455        0.00000000           3165.455
## 2           412.6667        0.08333333           3742.417
## 3           534.1000        0.00000000           3682.700
##   ENSG00000232439.1 ENSG00000199710.1 ENSG00000270777.1 ENSG00000099953.9
## 1                 0                 0         0.1818182          28.18182
## 2                 0                 0         0.0000000           5.75000
## 3                 0                 0         0.6000000          21.70000
##   ENSG00000201440.1 ENSG00000251313.1 ENSG00000223581.1 ENSG00000239650.4
## 1                 0        0.00000000                 0          7.454545
## 2                 0        0.08333333                 0          9.833333
## 3                 0        0.00000000                 0         19.500000
##   ENSG00000143061.17 ENSG00000276929.1 ENSG00000223869.1 ENSG00000258537.5
## 1           1029.818                 0                 0        0.09090909
## 2           1173.083                 0                 0        0.08333333
## 3            258.400                 0                 0        0.00000000
##   ENSG00000276070.4 ENSG00000273175.1 ENSG00000240458.1 ENSG00000228136.1
## 1         0.3636364          2.636364                 0                 0
## 2         0.0000000          1.416667                 0                 0
## 3         0.0000000          0.400000                 0                 0
##   ENSG00000261325.1 ENSG00000248415.1 ENSG00000232922.1 ENSG00000255700.2
## 1        0.09090909          2.545455               0.0                 0
## 2        0.08333333          2.333333               0.5                 0
## 3        0.00000000          3.200000               0.1                 0
##   ENSG00000234828.8 ENSG00000237300.2 ENSG00000083817.8 ENSG00000227409.1
## 1              0.00        0.09090909          393.7273                 0
## 2              0.25        0.00000000          165.0833                 0
## 3              5.20        0.00000000          230.5000                 0
##   ENSG00000175985.9 ENSG00000115850.9 ENSG00000234145.1 ENSG00000226080.1
## 1          3.272727         0.4545455         0.9090909         0.1818182
## 2         11.250000         2.0000000         1.9166667         0.0000000
## 3          9.600000         2.5000000         1.5000000         0.0000000
##   ENSG00000240419.1 ENSG00000260657.2 ENSG00000264801.1 ENSG00000207087.1
## 1                 0         0.4545455         21.000000                 0
## 2                 0         0.9166667          9.833333                 0
## 3                 0         0.4000000         11.800000                 0
##   ENSG00000265799.1 ENSG00000279333.1 ENSG00000211591.1 ENSG00000278598.1
## 1         0.4545455         0.7272727                 0                 0
## 2         0.0000000         1.1666667                 0                 0
## 3         0.1000000         2.3000000                 0                 0
##   ENSG00000205643.10 ENSG00000234052.1 ENSG00000243156.7 ENSG00000226876.2
## 1           94.18182                 0          1974.182                 0
## 2          286.25000                 0          2390.333                 0
## 3          150.20000                 0          2190.000                 0
##   ENSG00000082175.14 ENSG00000183629.13 ENSG00000145476.15
## 1          0.2727273         27.7272727           583.9091
## 2          0.7500000          0.4166667           496.7500
## 3          0.0000000         15.4000000          1138.3000
table(km$cluster)
## 
##  1  2  3 
## 11 12 10
plot_heatmap(km)

# Record sessionInfo()

The sessionInfo() prints version information about R and any attached packages. It’s a good practice to always run this command at the end of your R session and record it for the sake of reproducibility in the future.

sessionInfo()
## R version 3.5.1 (2018-07-02)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## Running under: Windows 10 x64 (build 17134)
## 
## Matrix products: default
## 
## locale:
## [1] LC_COLLATE=English_United States.1252 
## [2] LC_CTYPE=English_United States.1252   
## [3] LC_MONETARY=English_United States.1252
## [4] LC_NUMERIC=C                          
## [5] LC_TIME=English_United States.1252    
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] bindrcpp_0.2.2 tidyr_0.8.2    tibble_1.4.2   dplyr_0.7.8   
## [5] readr_1.3.0    ggplot2_3.1.0  knitr_1.21    
## 
## loaded via a namespace (and not attached):
##  [1] Rcpp_1.0.0       pillar_1.3.1     compiler_3.5.1   plyr_1.8.4      
##  [5] bindr_0.1.1      tools_3.5.1      digest_0.6.18    jsonlite_1.6    
##  [9] evaluate_0.12    gtable_0.2.0     pkgconfig_2.0.2  rlang_0.3.0.1   
## [13] yaml_2.2.0       xfun_0.4         withr_2.1.2      stringr_1.3.1   
## [17] hms_0.4.2        grid_3.5.1       tidyselect_0.2.5 glue_1.3.0      
## [21] R6_2.3.0         rmarkdown_1.11   purrr_0.2.5      magrittr_1.5    
## [25] codetools_0.2-15 scales_1.0.0     htmltools_0.3.6  assertthat_0.2.0
## [29] colorspace_1.3-2 labeling_0.3     stringi_1.2.4    lazyeval_0.2.1  
## [33] munsell_0.5.0    crayon_1.3.4